This Project focuses on pharmacologic plasticity in two key classes of analgesics used in the treatment of chronic pain: alpha 2-adrenergic and opioid agonists. Clonidine is approved in the treatment of neuropathic cancer pain, and animal models demonstrate an increase in potency (and perhaps efficacy) of alpha2-adrenergic agonists in hypersensitivity states following injury to the peripheral and central nervous systems. We have noted that alpha2-adrenergic agonists produce analgesia in part by stimulating spinal cholinergic activity. Following nerve injury this component becomes dominant, and blocking spinal cholinergic receptors completely antagonizes the anti-hypersensitivity effects of alpha2-adrenergic agonists. Other data demonstrate that this increased reliance on cholinergic systems is related to generation of nitric oxide in the spinal cord. The first two aims of this Project, therefore, are to investigate the causes of this shift in apparent circuitry in chronic pain. Specifically, we will test that pharmacologic plasticity following peripheral nerve injury reflects: 1. Alterations in noradrenergic innervation and alpha2-adrenergic receptor number, location, or function 2. Increased reliance of alpha2-adrenergic agonist effect on spinal cholinergic activity and nitric oxide synthesis An even more marked change accompanies the generation of hypersensitivity pain states in animals: spinally administered opioids completely lose their effect (except, perhaps, fully efficacious agents like DAMGO), and systemically administered opioids have diminished potency and, in some cases, efficacy. We will build on preliminary observations that link spinal opioid receptor activation to spinal release of adenosine, which mediates specifically reduction in hypersensitivity, and test the hypothesis that pharmacologic plasticity following peripheral nerve injury reflects: 3. Disruption of opioid-induced adenosine receptor activation A variety of methods, from behavior to location and expression of specific protein and mRNA species will be employed, and will be complemented by two trials in Project V.